Reactive Microneedle Patches with Antibacterial and Dead Bacteria‐Trapping Abilities for Skin Infection Treatment

Abstract Bacterial skin infections are highly prevalent and pose a significant public health threat. Current strategies are primarily focused on the inhibition of bacterial activation while disregarding the excessive inflammation induced by dead bacteria remaining in the body and the effect of the acidic microenvironment during therapy. In this study, a novel dual‐functional MgB2 microparticles integrated microneedle (MgB2 MN) patch is presented to kill bacteria and eliminate dead bacteria for skin infection management. The MgB2 microparticles not only can produce a local alkaline microenvironment to promote the proliferation and migration of fibroblasts and keratinocytes, but also achieve >5 log bacterial inactivation. Besides, the MgB2 microparticles effectively mitigate dead bacteria‐induced inflammation through interaction with lipopolysaccharide (LPS). With the incorporation of these MgB2 microparticles, the resultant MgB2 MN patches effectively kill bacteria and capture dead bacteria, thereby mitigating these bacteria‐induced inflammation. Therefore, the MgB2 MN patches show good therapeutic efficacy in managing animal bacterial skin infections, including abscesses and wounds. These results indicate that reactive metal borides‐integrated microneedle patches hold great promise for the treatment of clinical skin infections.


H 2 O 2 scavenging test
H 2 O 2 can react with Ti(SO 4 ) 2 to generate the yellow complex with a characteristic absorption peak at 405 nm.H 2 O 2 (100 μL, 0.3 M) was incubated with MgB 2 MPs (100 μL, final concentration: 80, 160, and 320 μg/mL) at 37°C for 5 h.The 150 μL supernatant solution was collected by removing MgB 2 MPs and then mixed with 50 μL Ti(SO 4 ) 2 solution (6 mg/mL).Their absorbance was measured at the wavelength of 405 nm using the microplate reader.

•OH scavenging test
•OH scavenging activity test was studied by using salicylic acid.The salicylic acid can react with •OH to form the pink complex with a characteristic absorption at 510 nm.The work solutions containing H 2 O 2 (100 μL, 50 mM) and FeSO 4 (2 mM, 100 μL) were co-incubated for 10 min.MgB 2 MPs (final concentration: 80, 160, and 320 μg/mL) were added into the mixed work reaction and co-incubated for 15 min.The salicylic acid (4 mM, 100 μL) was added into the above solution (100 μL) after removing MgB 2 MPs.Their absorbance was measured at the wavelength of 510 nm using the microplate reader.

The pH variation of MgB 2 MPs hydrolysate
The 3 mL MgB 2 MPs solution (0-320 μg/mL) containing sodium citrate/citric acid buffer (pH 7.5 or 5.5) and performed in the shaker (200 rpm) at 37°C.After 24 h, the pH values of various solutions were detected by a pH meter.

Cytotoxicity assay
The cytotoxicity of MgB 2 MPs was evaluated using HEK293 cells.HEK293 cells were incubated with MgB 2 MPs (final concentration: 0-160 μg/mL) in the 96-well plate.Their cytotoxicity was evaluated after incubation at 37°C for 24 h by a LDH assay.

Antibacterial performance of MgB 2 MN
The antibacterial performance of MgB 2 MN was studied by the CFU counting method.
The bacterial suspensions (10 6 CFU/mL, 200 μL) containing MgB 2 MN were incubated in a shaker (shaking speed: 200 rpm) at 37°C.At regular intervals, the ten microliters of bacterial suspensions were serially diluted using saline and plated on LB agar plates.Finally, the number of colonies were counted.

The pH variation of MgB 2 MN hydrolysate
Sodium citrate/citric acid buffer solutions containing MgB 2 MNs (pH 7.5, 3 mL) and performed in the shaker (200 rpm) at 37°C.At regular intervals, their pH values were monitored using the pH meter.

Dead bacteria-trapping ability of MgB 2 MN
The dead bacteria (10 8 CFU/mL, 1 mL) containing MgB 2 MN were incubated at 37°C for 24 h.MNs were fixed with glutaraldehyde (2.5%) for 12 h at 4°C, then were performed sequential dehydrating treatments using the gradient ethanol solutions.The empty MNs without MgB 2 MPs as the control.The final MNs were observed by SEM after sputter-coating 5 nm gold.

Mechanical strength of MgB 2 MN
The force sensor was fastened above the fixed station and approached the MgB 2 MN at the speed of 0.2 mm/min.The force measurements began when the sensor first touched the MN tips and ended when the sensor traveled 0.2 mm.

Wound and skin insertion test
The rhodamine-stained MgB 2 MNs were inserted into the living mice skin and wound for 5 min to examine their penetration capability.MN insertion sites in wounds were observed by using an Olympus FV3000 confocal laser scanning microscope (CLSM).
MN insertion sites in skin were observed by Hematoxylin and eosin (H&E) staining.

Cell proliferation assay
The keratinocytes (HaCat) and fibroblasts (3T3) were used in the proliferation assay.
HaCat and 3T3 cells (10 5 cells) were incubated with MgB 2 MNs at 37°C for 48 h.The cell viability was evaluated by using a cck-8 assay.

Cell migration assay
The migration of fibroblasts and keratinocytes was studied by using the scratch assay.
HaCat and 3T3 cells were plated into a 6-well plate to reach confluence, and then were scratched by MgB 2 MN and the empty MN (control).The images of cells were taken at 0 and 48 h using an Olympus inverted microscope.The female Balb/c mice (18-22 g, Qinglong Mountain Company, China) were used to build the infected subcutaneous abscess model.We removed the dorsal hair of the mice.The 100 μL MRSA (OD = 1) were inoculated into the right side of spine on the back.After 1 day, the ten mice were randomly divided into two groups.These patches were inserted into MRSA infected subcutaneous abscess by hand and remained for 24 h.The subcutaneous abscess was treated with MNs on 8 days.At 8-day treatment, the bacteria infected skin tissues were harvested for pH measurement.At 16-day treatment, the bacteria infected skin tissues were harvested for histological analysis, counting bacterial CFU, and then the levels of TNF-α, iNOS, IL-1β, and IL-6 were measured via QPCR.

Wound infection model
The round wounds (~6 mm) were constructed and then infected (10 7 CFU MRSA) on the back of mice, all infected wounds were finally covered with a special kind of plastic sticker (Tegaderm Film, 3M, 1624W).After 24 h, the ten mice were randomly divided into two groups.These patches were inserted into MRSA infected wounds using our hand and remained on wounds for 24 h.All wounds were treated with MNs on 3 days.On day 9, the wounded skin was harvested for counting bacterial CFU, histological analysis, and then the levels of TNF-α, iNOS, IL-1β, and IL-6 were measured via QPCR.

Dead bacteria induced wound inflammation
The round wounds (~6 mm) were constructed and then infected (10 7 CFU HIB) on the back of mice, all infected wounds were finally covered with a special kind of plastic sticker (Tegaderm Film, 3M, 1624W).After 1 day, the 10 mice were randomly divided into 2 groups.The pure wounds without infection in other ten mice were used as the control groups.The HIB-induced wounds showed an acute exudative state before MNs treatment.The pure wounds showed a crusted and dry state before MNs treatment.The wounds were treated with MNs on 3 days.On day 12, the wounded skin was harvested for the histological analysis.

Histological analysis
The infected tissues or major organs (heart, liver, spleen, lung and kidney) were dissected, fixed in 4% paraformaldehyde solution, paraffined, sectioned, and then analyzed by H&E staining.

Reproducibility and statistical analysis
All quantitative data in each experiment were repeated and evaluated from three independent experiments with similar results.The data were performed as mean ± SD.
animals were handled in accordance with the Guidelines for the Protection and Use of Laboratory Animals of the National Institutes of Health and with the approval from the Ethical Review Committee of Nanjing Drum Tower Hospital.The animal experiments were conducted with the Animal Investigation Ethics Committee of The Affiliated Drum Tower Hospital of Nanjing University Medical School (2023AE02009).
Figure S1.SEM image of bulk MgB 2 materials.

Figure S6 .
Figure S6.(A) CFU counts and (B) the inactivation efficiency of MRSA treated by Mg 2+ with different concentrations for 1 h.

Figure S7 .
Figure S7.(A) CFU counts and (B) the inactivation efficiency of MRSA after incubation under different pH condition for 1 h.

Figure S8 .
Figure S8.Mechanical property of the MgB 2 MN under normal compressive load.

Figure S10 .
Figure S10.H&E-stained section of mouse skin showing the indents caused by the penetration of a single microneedle.

Figure S13 .
Figure S13.SEM image of MgB 2 MN after incubation in saline for 24 h.

Figure S14 .
Figure S14.Treatment of dead bacteria-induced wound inflammation.(A) Photographs of dead bacteria-induced wound inflammation treated with or without MgB 2 MN.(B) H&E staining of HIB (dead MRSA)-induced wounds.Immunofluorescence staining with the (C) macrophage cell marker F4/80 and (D) neutrophil cell marker myeloperoxidase (MPO) of HIB-induced wounds.